Neural basis for improgan antinociception.

Improgan, the prototype compound of a novel class of non-opioid analgesic drugs derived from histamine antagonists, attenuates thermal and mechanical nociception in rodents following intracerebroventricular (i.c.v.) administration. Improgan does not bind to known opioid, histamine or cannabinoid receptors, and its molecular target has not been identified. It is known however, that improgan acts directly in the periaqueductal gray and the rostral ventromedial medulla to produce its antinociceptive effects, and that inactivation of the rostral ventromedial medulla prevents the antinociceptive effect of improgan given i.c.v. Here we used in vivo single-cell recording in lightly anesthetized rats to show that improgan engages pain-modulating neurons in the medulla to produce antinociception. Following improgan administration, OFF-cells, which inhibit nociception, became continuously active and no longer paused during noxious stimulation. The increase in OFF-cell firing does not represent a non-specific neuroexcitant effect of this drug, since ON-cell discharge, associated with net nociceptive facilitation, was depressed. NEUTRAL-cell firing was unaffected by improgan. The net response of rostral ventromedial medulla (RVM) neurons to improgan is thus comparable to that evoked by mu-opioids and cannabinoids, well known RVM-active analgesic drugs. This common basis for improgan, opioid, and cannabinoid antinociception in the RVM supports the idea that improgan functions as a specific analgesic agent.

Antinociceptive activity of furan-containing congeners of improgan and ranitidine.

Furan-containing congeners of the histamine H(2) receptor antagonist ranitidine were synthesized and tested for improgan-like antinociceptive activity. The most potent ligand of the series, VUF5498, is the most potent improgan-like agent described to date (ED(50)=25 nmol, icv). This compound is approximately equal in potency with morphine. These non-imidazole, improgan-like pain relievers further define the structural requirements for analgesics of this class and are important tools for ongoing mechanism-based studies.

Actions of tacrine and galanthamine on histamine-N-methyltransferase.

Histamine-synthesizing neurons in the brain may play an important role in cognition, and a histaminergic deficit has been found in Alzheimer's disease (AD). The AD medication tacrine was previously shown to inhibit some forms of rodent histamine-N-methyltransferase (HNMT), but the effects of AD drugs have not been investigated on human HNMT activity. Presently, the effects of tacrine and galanthamine (another AD medication) were studied on the activity of several forms of human and rat HNMT. Tacrine (0.01-10 microM) inhibited both human and rat HNMT activity in a concentration-dependent manner, but was less potent on both human embryonic kidney and recombinant human brain HNMT than on rat kidney HNMT (IC50 values were 0.46 and 0.70 microM vs. 0.29 microM, respectively). Galanthamine (up to 10 microM) did not influence the activity of rat kidney or human HNMT. Tacrine, but not galanthamine, may achieve brain levels sufficient to influence histamine metabolism in some patients treated for AD.

Absence of 5-HT3 and cholinergic mechanisms in improgan antinociception.

Improgan, an analgesic derived from histamine antagonists, acts in the brain stem to activate descending non-opioid, pain-relieving circuits, but the mechanism of action of this drug remains elusive. Because improgan has a moderate affinity for 5-HT(3) receptors, and, since cholinergic and serotonergic drugs can modulate descending analgesic circuits, roles for 5-HT(3), nicotinic and muscarinic receptors in improgan antinociception were presently investigated in rats. Improgan (80 microg, icv) induced nearly maximal inhibition of hot plate and tail flick nociceptive responses, and these actions we unaffected by antagonists of muscarinic (atropine, 5.9 mg/kg, i.p.) and nicotinic (mecamylamine, 2 mg/kg, i.p.) receptors. Control experiments verified that these antagonist treatments were maximally effective against muscarinic and nicotinic antinociception in both tests. In addition, improgan antinociception was unaffected by icv pretreatment with a 5-HT(3) antagonist (ondansetron, 20 microg). When given alone, icv treatment with neither this antagonist nor a 5-HT(3) agonist (m-chlorophenylbiguanide, 1000 nmol, icv) modified thermal nociceptive latencies. These results show no role for supraspinal cholinergic and 5-HT(3) receptors in improgan antinociception. The findings help to narrow the search for the relevant mediators of the action of this novel analgesic agent.

Activation of brain stem nuclei by improgan, a non-opioid analgesic.

Improgan is a compound developed from histamine antagonists which shows the pre-clinical profile of a highly effective, non-opioid analgesic when administered into the rodent CNS. Pharmacological studies suggest that improgan activates descending pain-relieving circuits, but the brain and spinal sites of action of this drug have not been previously studied. Presently, the effects of intracerebral and intrathecal microinjections of improgan were evaluated on thermal nociceptive responses in rats. Improgan produced large, dose- and time-related reductions in nociceptive responses following administration into the ventrolateral periaqueductal gray (PAG), the dorsal PAG, and the rostral ventromedial medulla (RVM). The drug had no measurable effects after injections into the caudate nucleus, basolateral amygdala, hippocampus, ventromedial hypothalamus, superior colliculi, ventrolateral medulla, or the spinal subarachnoid space. Inactivation of the RVM by muscimol microinjections completely attenuated antincociceptive responses produced by intraventricular improgan. These findings, taken with earlier results, show that, like opioids and cannabinoids, improgan acts in the PAG and RVM to activate descending analgesic systems. Unlike these other analgesics, improgan does not act in the spinal cord or in CNS areas outside of the brain stem.

Inhibition of improgan antinociception by the cannabinoid (CB)(1) antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A): lack of obligatory role for endocannabinoids acting at CB(1) receptors.

Improgan, a nonopioid antinociceptive agent, activates descending, pain-relieving mechanisms in the brain stem, but the receptor for this compound has not been identified. Because cannabinoids also activate nonopioid analgesia by a brain stem action, experiments were performed to assess the significance of cannabinoid mechanisms in improgan antinociception. The cannabinoid CB(1) antagonist N-(piperidin-1-yl)-5-(4-chloro phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) induced dose-dependent inhibition of improgan antinociception on the tail-flick test after i.c.v. administration in rats. The same treatments yielded comparable inhibition of cannabinoid [R-(+)-(2,3-dihydro-5-methyl-3-[(4-mor pholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl)methanone monomethanesulfonate, WIN 55,212-2] analgesia. Inhibition of improgan and WIN 55,212-2 antinociception by SR141716A was also observed in Swiss-Webster mice. Radioligand binding studies showed no appreciable affinity of improgan on rat brain, mouse brain, and human recombinant CB(1) receptors, ruling out a direct action at these sites. To test the hypothesis that CB(1) receptors indirectly participate in improgan signaling, the effects of improgan were assessed in mice with a null mutation of the CB(1) gene with and without SR141716A pretreatment. Surprisingly, improgan induced complete antinociception in both CB(1) (-/-) and wild-type control [CB(1) (+/+)] mice. Furthermore, SR141716A inhibited improgan antinociception in CB(1) (+/+) mice, but not in CB(1) (-/-) mice. Taken together, the results show that SR141716A reduces improgan antinociception, but neither cannabinoids nor CB(1) receptors seem to play an obligatory role in improgan signaling. Present and previous studies suggest that Delta(9)-tetrahydrocannabinol may act at both CB(1) and other receptors to relieve pain, but no evidence was found indicating that improgan uses either of these mechanisms. SR141716A will facilitate the study of improgan-like analgesics.

Extracellular histamine levels in the feline preoptic/anterior hypothalamic area during natural sleep-wakefulness and prolonged wakefulness: an in vivo microdialysis study.

Increased activity of the histaminergic neurons of the posterior hypothalamus has been implicated in the facilitation of behavioral wakefulness. Recent evidence of reciprocal projections between the sleep-active neurons of the preoptic/anterior hypothalamus and the histaminergic neurons of the tuberomammillary nucleus suggests that histaminergic innervation of the preoptic/anterior hypothalamic area may be of particular importance in the wakefulness-promoting properties of histamine. To test this possibility, we used microdialysis sample collection in the preoptic/anterior hypothalamic area of cats during natural sleep-wakefulness cycles, 6 h of sleep deprivation induced by gentle handling/playing, and recovery sleep. Samples were analyzed by a sensitive radioenzymatic assay. Mean basal levels of histamine in microdialysate during periods of wakefulness (1.155+/-0.225 pg/microl) did not vary during the 6 h of sleep deprivation. However, during the different sleep states, dramatic changes were observed in the extracellular histamine levels of preoptic/anterior hypothalamic area: wakefulness>non-rapid eye movement sleep>rapid eye movement sleep. Levels of histamine during rapid eye movement sleep were lowest (0.245+/-0.032 pg/microl), being significantly lower than levels during non-rapid eye movement sleep (0.395+/-0.081 pg/microl) and being only 21% of wakefulness levels. This pattern of preoptic/anterior hypothalamic area extracellular histamine levels across the sleep-wakefulness cycle closely resembles the reported single unit activity of histaminergic neurons. However, the invariance of histamine levels during sleep deprivation suggests that changes in histamine level do not relay information about sleep drive to the sleep-promoting neurons of the preoptic/anterior hypothalamic area.

A role for spinal, but not supraspinal, alpha(2) adrenergic receptors in the actions of improgan, a powerful, non-opioid analgesic.

Improgan is a derivative of cimetidine that induces non-opioid antinociception after intracerebroventricular (i.c.v.) administration, but the mechanism of action of this compound remains unknown. Since activation of either supraspinal or spinal alpha(2) adrenergic receptors can induce antinociception, and since improgan showed affinity for these receptors in vitro, the effects of the alpha(2) antagonist yohimbine on improgan antinociception were presently studied in rats on the hot plate and tail flick tests. Systemic yohimbine pretreatment (4 mg/kg, i.p.) completely blocked improgan antinociception (80 microg, i.c.v.), suggesting a mediator role for alpha(2) receptors. However, i.c.v. pretreatment with yohimbine (30 microg) had no effect on improgan antinociception. Since this treatment completely antagonized clonidine antinociception (40 microg, i.c.v.), supraspinal alpha(2) receptors seem to mediate the antinociceptive effects of clonidine, but not that produced by improgan. In contrast, intrathecal (i.t.) yohimbine pretreatment (30 microg) completely blocked the antinociception elicited by i.c.v. improgan and i.c.v. morphine. These results suggest that spinal (but not supraspinal) alpha(2) adrenergic receptors play a significant role in the pain-relieving actions of improgan. Furthermore, although improgan shows some affinity at alpha(2) receptors, this drug does not act directly at these receptors to induce antinociceptive responses. Like several other classes of analgesics, improgan-like drugs seem to activate non-opioid, descending pain-relieving circuits.

Significance of GABAergic systems in the action of improgan, a non-opioid analgesic.

Improgan is the prototype drug from a new class of non-opioid analgesics chemically related to histamine and histamine antagonists, but the mechanism of action of these compounds has not been identified. Because several classes of analgesics act in the brain by reducing GABAergic inhibition of endogenous pain-relieving circuits, and because the activity of these substances is abolished by the GABA(A) agonist muscimol, the present study assessed the effects of muscimol on improgan antinociception in rats. Intracerebroventricular (icv) improgan (80 microg) and morphine (20 microg) both induced 80-100% of maximal analgesic responses on the tail flick test 10 to 30 min later. However, muscimol pretreatment (0.5 microg, icv) completely eliminated the antinociceptive activity of both compounds. Since improgan in vitro lacks activity at opioid and GABA(A) receptors, these findings: 1) confirm earlier literature showing that muscimol inhibits morphine analgesia, and 2) suggest that improgan activates a supraspinal, descending analgesic pathway, possibly via inhibition of GABAergic transmission. Since muscimol is the first compound discovered which inhibits improgan analgesia, muscimol will be a useful tool for the further characterization of this new class of pain-relieving substances.

Improgan, a cimetidine analog, induces morphine-like antinociception in opioid receptor-knockout mice.

Improgan is an analog of the H(2) antagonist cimetidine that does not act on known histamine receptors, but induces highly effective analgesia in rodents following intracerebroventricular (icv) administration. Since the mechanism of action of this compound remains unknown, improgan analgesia was characterized presently with the tail immersion nociceptive test in mutant mice lacking either the mu (exon 1 of MOR-1), delta (exon 2 of DOR-1) or kappa (exon 3 of KOR-1) opioid receptor. Improgan (30 microg, icv) induced reversible, maximal analgesia in both sexes of all three genotypes (+/+, +/- and -/-) of MOR-1 mutant mice 10 and 20 min after administration, whereas morphine analgesia was reduced (+/-) or abolished (-/-) in these subjects. In DOR-1 mutant mice, improgan was equally effective in all three genotypes, despite the reduction (+/-) or complete loss (-/-) of delta opioid receptor (3H-[D-Pen(2), D-Pen(5)]enkephalin, DPDPE) binding. Similarly, improgan analgesia was equivalent in all three genotypes of KOR-1 mutant mice, whereas kappa-mediated analgesia (U50,488) and kappa opioid (3H-U69,593) binding were abolished in the homozygous (-/-) mice. These studies demonstrate that improgan analgesia does not require intact MOR-1, DOR-1, or KOR-1 genes, and support the hypothesis that improgan-like analgesics act in the CNS by non-opioid mechanisms.

A third life for burimamide. Discovery and characterization of a novel class of non-opioid analgesics derived from histamine antagonists.

Burimamide, a histamine (HA) derivative with both H2- and H3-blocking properties, induces antinociception when injected into the rodent CNS. Several related compounds share this property, and structure-activity studies have shown that this new class of analgesics is distinct from known HA antagonists. The prototype, named improgan, shows a preclinical profile of a highly effective analgesic, with activity against thermal, mechanical and inflammatory nociception after doses that do not alter motor balance or locomotor activity. Improgen analgesia is not blocked by opioid antagonists and is observed in opioid receptor knock-out mice. Unlike morphine, improgan does not induce tolerance after daily dosing. Extensive in vitro pharmacology studies have excluded known histaminergic, opioid, serotonergic, GABAergic and adrenergic receptor mechanisms, as well as 50 other sites of action. The improgan-like analgesic activity of some HA congeners suggests an analgesic action on a novel HA receptor, but further studies are required to substantiate this. Studies in progress are characterizing the sites and mechanisms of action of improgan, and developing brain-penetrating derivatives that could be useful for clinical pain.

Pharmacokinetic characterization of the indole alkaloid ibogaine in rats.

To investigate the pharmacokinetic properties of ibogaine, a putatively anti-addictive alkaloid, levels of this drug were quantified in plasma and tissues for up to 3 h following i.v. infusion in rats. Immediately following a 31-35 min infusion (20 mg/kg), mean plasma ibogaine levels were 373 ng/ml; these values declined rapidly thereafter in a biexponential manner. The plasma time course in 5 of 7 animals demonstrated an excellent fit to a two-compartment pharmacokinetic model, with alpha and beta half-lives of 7.3 min and 3.3 h, respectively. Drug clearance was estimated to be 5.9 l/h (n = 7). Ibogaine levels in brain, liver and kidney 3 h after the end of drug infusion were 143-170 ng/g, close to simulated values for the peripheral pharmacokinetic compartment. However, 3-h drug levels in adipose tissue were much higher (3,328 ng/g), implying the need for a more complex pharmacokinetic model. Mechanisms for the initial, rapid disappearance of plasma ibogaine are thought to include metabolic demethylation as well as redistribution to body stores. The sequestration of ibogaine by adipose tissue probably contributes to a protracted persistence of drug in the body. This persistence may be underestimated by the beta half-life reported in the present study.

Increased histamine release and granulocytes within the thalamus of a rat model of Wernicke's encephalopathy.

The current study examined the possible role of increased histamine release and granulocyte activity in the vascular changes that precede the onset of necrotic lesions with the thalamus of the pyrithiamine-induced thiamine deficiency (PTD) rat model of Wernicke's encephalopathy (WE). An increase in histamine release and the number of granulocytes was observed in lateral thalamus on day 9 and in medial thalamus on day 10 of PTD treatment, a duration of thiamine deficiency associated with perivascular edema in this brain region. Within the hippocampus, histamine release was significantly increased on day 9, declined to control levels on days 10-12, and was significantly elevated on days 12-14. No granulocytes were observed in hippocampus of either PTD or control rats. These observations suggest that the release of histamine from nerve terminals and histamine and other vasoactive substances from granulocytes may be responsible for thiamine deficiency-induced vascular breakdown and perivascular edema within thalamus.

Antinociceptive activity of derivatives of improgran and burimamide.

Improgan, a compound related to H2 and H3 antagonists, induces antinociception in rodents after intraventricular administration. Characteristics of improgan and its congeners include: (a) morphine-like antinociception on thermal and mechanical tests in two species; (b) no impairment of motor coordination or locomotor activity; (c) evidence for a novel, nonopioid mechanism that is independent of known histamine receptors; (d) lack of tolerance with daily dosing; and (e) unique structure-activity relationships (SARs). Presently, the antinociceptive activity of several new derivatives of improgan was investigated in rats. Among compounds similar to burimamide, VUF4577 (possessing a two-carbon side chain) and VUF4582 (an N-phenyl derivative of VUF4577) induced complete, dose- and time-dependent antinociception on the hot-plate and tail-flick tests with no behavioral side effects. These compounds (with ED50 values of 71-117 nmol) were approximately twice as potent as burimamide itself (a four-carbon derivative). Two other derivatives in which the thiourea group (C=S, known to cause human toxicity) was replaced by either nitroethene (C=CH-NO2, VUF5405) or urea (C=O, VUF5407) also showed effective, potent antinociception on both assays. The latter compound is the most potent improgan-like drug discovered to date (ED50 = 71 nmol). Furthermore, positional isomers of antinociceptive compounds either lacked activity (VUF5394) or induced toxicity (VUF5393), revealing a high degree of pharmacological specificity. Although the mechanism of improgan antinociception remains unknown, the present results show promise for the further development of safe, effective, and potent pain-relieving compounds.

Antinociceptive activity of impentamine, a histamine congener, after CNS administration.

The brain neuromodulator histamine induces antinociception when administered directly into the rodent CNS. However, several compounds derived from H2 and H3 antagonists also produce antinociception after central administration. Pharmacological studies have shown that a prototype of these agents, improgan, induces analgesia that is not mediated by actions on known histamine receptors. Presently, the antinociceptive properties of a compound that chemically resembles both improgan and histamine were investigated in rats. Intraventricular (i.v.t.) administration of impentamine (4-imidazolylpentylamine) induced reversible, near-maximal antinociception on the hot plate and tail flick tests (15 microg, 98 nmol). The dose-response function was extremely steep, however, since other doses showed either no effect or behavioral toxicity. On the tail flick test, impentamine antinociception was resistant to antagonism by blockers of H1, H2, or H3 receptors, similar to characteristics previously found for improgan. In contrast, histamine antinociception was highly attenuated by H1 and H2 antagonists. These findings suggest that: 1) the histamine congener impentamine may induce antinociception by a mechanism similar to that produced by improgan, and 2) additional histamine receptors may be discovered that are linked to pain-attenuating processes.

Effects of selected histamine H3 receptor antagonists on tele-methylhistamine levels in rat cerebral cortex.

The H3 antagonist thioperamide is thought to act on brain H3 autoreceptors to increase both the release and metabolism of neuronal histamine (HA). Our studies investigated the effects of several new brain-penetrating H3 antagonists on rat cerebral cortical levels of the HA metabolite tele-methylhistamine (t-MH). Animals were pretreated with H3 antagonists (0.3 to 30 mg/kg; 1-4 hr; i.p.) in the presence or absence of the monoamine oxidase inhibitor pargyline to prevent metabolism of t-MH. Cortical t-MH levels were measured by both radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS). Pargyline (60 mg/kg; 1 hr; i.p.) produced an approximately 2-fold increase in t-MH levels as measured by either GC-MS or RIA. Thioperamide (+/- pargyline) increased t-MH levels as measured by both GC-MS and RIA. In contrast, neither 5-cyclohexyl-1-(4-imidazol-4-ylpiperidyl)pentan-1-one (GT-2016) (+/- pargyline), 4-(6-cyclohexylhex-cis-3-enyl)imidazole (GT-2227) (+/- pargyline), nor clobenpropit (minus pargyline) increased t-MH levels as measured by GC-MS. A good agreement was found between t-MH levels as determined by either RIA or GC-MS except after treatment with GT-2016, which increased apparent t-MH brain levels according to the former but not the latter method. Subsequent studies suggest the in vivo formation of a GT-2016 metabolite, which can cross-react in the t-MH RIA. Although all H3 receptor antagonists studied to date seem capable of enhancing brain HA release, only thioperamide presently was found to enhance cortical t-MH levels. Thus, H3 receptor antagonists may differentially affect HA release and turnover, and brain t-MH levels may not be reliable predictors of H3 agonist, partial agonist, or antagonist in vivo activity.

Absence of antinociceptive tolerance to improgan, a cimetidine analog, in rats.

Improgan, an analog of the histamine receptor antagonist cimetidine, produces highly effective analgesia following intraventricular injection. The present study examined changes in the antinociceptive effects of improgan following once daily intraventricular injections. Improgan (100-150 microg) produced near maximal antinociception 10 and 30 min after daily administration on all 4 test days, whereas comparable morphine treatments (50 microg) induced considerable tolerance. Thus, improgan produced highly effective analgesia without the development of tolerance.

Sex differences in ibogaine antagonism of morphine-induced locomotor activity and in ibogaine brain levels and metabolism.

The present study demonstrates that the putative antiaddictive agent ibogaine produces more robust behavioral effects in female than in male rats and that these behavioral differences correlate with higher levels of ibogaine in the brain and plasma of female rats. There were no differences in basal locomotor activity between the sexes, and the response of rats to ibogaine differed between the sexes even in the absence of morphine. Five h after receiving ibogaine (40 mg/kg, i.p.). antagonism of morphine-induced locomotor activity was evident in female but not in male rats. Either 19 h after administration of ibogaine (10-60 mg/kg, i.p.), or one h after administration of noribogaine (5-40 mg/kg, i.p.), a suspected metabolite, antagonism of morphine was significantly greater in female than in male rats. Brain and plasma levels of ibogaine (1 h) and noribogaine (5 h), measured by gas chromatography-mass spectrometry, were greater in females as compared with males receiving the same dose of ibogaine. Levels of both ibogaine and noribogaine were substantially lower at 19 h than at earlier times after ibogaine administration, contrary to a previous study in humans. For both sexes, subcutaneous administration of ibogaine (40 mg/kg, i.p., 19 h) produced greater antagonism of morphine-induced locomotor activity than did a comparable intraperitoneal injection, consistent with previous studies from this laboratory demonstrating that the former route of administration produces higher levels of ibogaine in the brain. These data show that there are sex differences in the effects of ibogaine and that this may be due to decreased bioavailability of ibogaine in males as compared to females.